This invention is generally in the field of compositions and methods for the treatment and prevention of inflammatory responses involving P-selectin (formerly, GMP-140 or PADGEM) binding reactions, particularly adhesive interactions between platelets, leukocytes, and endothelial cells.
The adherence of leukocytes to vascular surfaces is a critical component of the inflammatory response, and is part of a complex series of reactions involving the simultaneous and interrelated activation of the complement, coagulation, and immune systems. Leukocyte adherence to vascular endothelium is a key initial step in migration of leukocytes to tissues in response to microbial invasion. The initial rolling contacts of leukocytes with the endothelium are mediated by the selectins, a family of receptors that interact with cell-surface carbohydrate ligands (reviewed in McEver, Curr. Opin Cell Biol., 4, pp 840-859 (1992); Lasky, Science, 258, pp. 964-969, (1992)). These transient adhesive interactions allow time for leukocytes to become activated by signaling molecules that are released from the endothelium or the underlying tissues. Upon activation, leukocytes functionally upregulate members of the integrin family of adhesion receptors. The integrins strengthen adhesion by binding to immunoglobulin-like counter-receptors on the endothelial cell (McEver, Curr. Opin Cell Biol., 4, pp 840-859 (1992)). Adhesion and signaling molecules function cooperatively to regulate leukocyte recruitment during the inflammatory response.
Leukocytes also adhere to activated platelets, through interactions of P-selectin on the activated platelet surface with carbohydrate ligands on the leukocyte surface (McEver, in Structure, Function, and Regulation of Molecules Involved in Leukocyte Adhesion, pp 135-150 (Lipsky et al., eds., Springer-Verlag, New York, 1993)). Platelet-leukocyte interactions may serve as important links between the hemostatic and inflammatory responses to tissue injury.
The coagulation and inflammatory pathways are regulated in a coordinated fashion in response to tissue damage. For example, in addition to becoming adhesive for leukocytes, activated endothelial cells express tissue factor on the cell surface and decrease their surface expression of thrombomodulin, leading to a net facilitation of coagulation reactions on the cell surface. In some cases, a single receptor can be involved in both inflammatory and coagulation processes. For example, the Mac-1 receptor on leukocytes, a member of the CD11-CD18 group, mediates phagocytosis and serves as a receptor for the degradation product of complement C3bi, is involved in one pathway of adherence of leukocytes to endothelium, mediates granulocyte aggregation, and binds coagulation Factor X.
Proteins involved in the hemostatic and inflammatory pathways are of interest for diagnostic purposes and treatment of human disorders. An example is P-selectin, formerly known as GMP-140 (granule membrane protein 140) or PADGEM, an integral membrane glycoprotein with an apparent molecular weight of 140,000 as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). P-selectin contains an N-terminal lectin-like domain, followed by an epidermal growth factor-like module, a series of consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a cytoplasmic tail, as described in the parent application, U.S. Ser. No. 07/320,408, filed Mar. 8, 1989, the teachings of which are incorporated herein. P-selectin is a member of the selectin family of adhesion receptors that mediate leukocyte interactions with vascular endothelium or platelets (McEver, Curr. Opin. Cell Biol., 4, pp. 840-849 (1992); Lasky, Science, 258, pp. 964-969 (1992); Bevilacqua and Nelson, J. Clin. Invest., 91, pp. 379-387 (1993)). The human P-selectin gene spans over 50 kilobases (kb) and contains 17 exons, most of which encode structurally distinct domains (Johnston et al., J. Biol. Chem., 265, pp. 21381-21385 (1990)).
P-selectin was first purified (as GMP-140) from human platelets by McEver and Martin, J. Biol. Chem., 259, pp. 9799-9804 (1984). Monoclonal and polyclonal antibodies to P-selectin were also prepared, as reported by McEver and Martin (1984) and P. E. Stenberg, et al., J. Cell Biol., 101, pp. 80-886 (1985). The protein is present in alpha granules of resting platelets but is rapidly redistributed to the plasma membrane following platelet activation, as reported by Stenberg, et al., (1985). The presence of P-selectin in endothelial cells and its biosynthesis by these cells was reported by McEver, et al., Blood, 70(5) Suppl. 1:355a, Abstract No. 1274 (1987). In endothelial cells, P-selectin is found in storage granules known as the Weibel-Palade bodies. When platelet or endothelial cells are activated by mediators such as thrombin, the membranes of the storage granules fuse with the plasma membrane, the soluble contents of the granules are released to the external environment, and membrane bound P-selectin is presented within seconds on the cell surface, where it mediates adhesion of neutrophils, monocytes, and subsets of lymphocytes (McEver, in Structure, Function, and Regulation of Molecules Involved in Leukocyte Adhesion, pp. 135-150 (Lipsky et al., eds., Springer-Verlag, New York, 1993)).
The expression of P-selectin, as observed by immunohistochemistry (McEver et al., J. Clin. Invest., 84, pp. 92-99 (1989)) and Northern blot analysis (Johnston et al., Cell, 56, pp. 1033-1044 (1989)), is restricted to megakaryocytes and endothelial cells. Under certain circumstances, steady-state levels of mRNA and protein are increased by inflammatory mediators such as tumor necrosis factor and endotoxin (Sanders et al., Blood, 80, pp. 795-800 (1992); Weller et al., J. Biol. Chem., 267, pp. 15176-15183 (1992); Hahne et al., J. Cell Biol., 121, pp. 655-664 (1993)). Thus, an understanding of the molecular mechanisms that control transcription of the P-selectin gene may help clarify the mechanisms for gene expression in megakaryocytes and endothelial cells and for regulation of leukocyte adhesion in response to tissue injury.
The promoters of several genes whose expression is restricted to endothelial cells or megakaryocytes have been partially characterized (Lee et al., J. Biol. Chem., 265, pp. 10446-10450 (1990); Wilson et al., Mol. Cell Biol., 10, pp. 4854-4862 (1990); Ravid et al., Mol. Cell Biol., 11, pp. 6116-6127 (1991); Romeo et al., Nature, 344, pp. 447-449 (1990); Uzan et al., J. Biol. Chem., 266, pp. 8932-8939 (1991)). The GATA element, initially recognized in erythroid-specific promoters, plays an important role in expression of some of these genes (Wilson et al., (1990), Romeo et al., (1990)). However, this element is not sufficient to mediate tissue-specific expression, as expression of the GATA-binding proteins does not directly correlate with expression of the genes containing GATA elements (Yamamoto et al., Genes & Dev., 4, pp. 1650-1662 (1990)). A functional ETS element has been identified in the megakaryocyte-specific gene for glycoprotein IIb (Prandini et al., J. Biol. Chem., 267, pp. 10370-10374 (1992); Lemarchandel et al., Mol. Cell. Biol., 13, pp. 668-676 (1993)), but this element is also found in genes expressed in other tissues (see e.g., Macleod et al., Trends Biochem. Sci., 17, pp. 251-256 (1992)).
It is an object of the present invention to characterize and provide DNA and RNA sequences of the 5' flanking region of the human P-selectin gene and to provide methods of using these sequences to specifically express P-selectin and other genes in endothelial cells and megakaryocytes.
It is another object of this invention to provide nucleic acid probes for screening for individuals with abnormal levels of P-selectin.
It is a further object of this invention to provide compositions and methods including the DNA or RNA sequences of the 5' flanking region of the human P-selectin gene for inhibiting or regulating P-selectin expression to control the inflammatory and hemostatic processes involving endothelial or megakaryocytic cells.